Abstract
Recent studies have established that the phenotype of epithelial stem cells residing in the corneal periphery (the limbus) depends on this niche’s distinct biomechanical properties. However, the signaling pathways underlying this dependency are still poorly understood. To address this issue, we investigated the effect of substrate stiffness on the migration, proliferation, and molecular phenotype of human limbal epithelial stem cells (LESCs). Specifically, we demonstrated that cells grown on collagen-based substrates with limbus-like compliance showed higher proliferation and stratification and lower migration capabilities, as well as higher levels of pro-proliferative markers Ki67 and β-Catenin, and LESC markers ΔNp63, ABCG2, and CK15. In contrast, cells on stiffer substrates lost these stem/progenitor cell markers, but instead expressed the key mechanotransduction factor YAP, as well as elevated levels of BMP4, a promotor of cell differentiation known to be negatively regulated by Wnt/β-Catenin signaling. This data allowed us to propose a new model that integrates the various molecular pathways involved in LESC response to substrate stiffness. This model will potentially be a useful guide to future research on the mechanisms underlying LESC loss following fibrosis-causing injuries.
Highlights
The cornea is the outermost tissue of the eye, and its transparency is determinant for maintaining vision quality
Collagen gels treated with collagenase or phosphate buffer saline (PBS) were transferred into Transwell culture inserts (Corning, Corning, NY, USA) and seeded with 1 × 105 limbal epithelial stem cells (LESCs) suspended in 1 mL of
To evaluate the effect of substrate compliance on LESC migration, cells were seeded onto half the area of three distinct culture surfaces, and imaged for 6 h by time-lapse microscopy (Figure 1b)
Summary
The cornea is the outermost tissue of the eye, and its transparency is determinant for maintaining vision quality. We recently demonstrated that the stiffening of the limbus matrix (e.g., due to chemical injury) promotes epithelial stem cell differentiation via mechanotransduction-dependent pathways [15], negatively impacting their renewal and leading to stem cell deficiency, corneal opacification, and vision loss. These findings highlighted the importance of corneal tissue biomechanics for maintaining epithelial homeostasis [13]. Our findings subsequently allowed us to propose a molecular model integrating probable signaling pathways involved in LESC mechanotransduction responses
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